First Report of Rhizome Rot on Ginger ( <i>Zingiber officinale</i> ) Caused by <i>Pseudomonas asiatica</i> in Shandong Province, China

Ginger (Zingiber officinale Rosc.) is an important economic spice crop in China, with 133,000 hectares concentrated primarily in Shandong Province. Between June and July 2024, bacterial rhizome rot was observed in ginger fields across Shandong Province with approximately 30% of plants exhibiting symptoms detected in the major production area of Weifang (GPS coordinates: 36°54'6.876" N, 118°51'17.003" E). Affected plants showed softening and rot of the basal roots and stems, along with water-soaked tissue degradation. The rhizomes were deteriorated, the leaves shown chlorosis, leading to rapid plant death. The surface of the infected plant parts was disinfected using a 1% NaClO solution for 1 minute, and cut into small sections using a sterile bistoury scalpel and streaked on nutrient agar (NA). Plates were incubated at 28℃. Bacterial colonies were subsequently isolated on NA using the streak plate technique. From 15 samples, pure colonies of seven distinct bacterial isolates were obtained. Pure cultures were then used for morphological, physiological, and biochemical characterization, as well as pathogenicity assay. Three isolates, designated as A13, A16, and A22, were selected for further analysis. On NA plate, the colonies appeared creamy white, moist, round, and smooth. The morphological characters were consistent with Pseudomonas spp. (Oller et al., 2025). These virulent isolates were characterized as facultatively anaerobic and Gram-negative. They demonstrated the ability to metabolize glucose, sorbitol, citrate, and mannitol. Additionally, they were positive for urease test and negative for indole, methyl red, and Voges-Proskauer tests. For the molecular identification, genomic DNA was extracted from pure cultures grown for 24 hours in nutrient broth medium. For strain A22, a representative isolate, the partial 16S rDNA, gapA, gyrB rpoD and gltA genes were amplified using primers 27F/1492R (Weisburg et al., 1991), gapA-F/gapA-R, gyrB-F/gyrB-R, PsEG30F/PsEG790R (Wei et al., 2023) and gltA+/gltA- (Michael et al., 2005), respectively. The GenBank accession numbers for these sequences are PV927154, SRR35933575, SRR35933566, SRR35933584 and SRR35933583, respectively. Phylogenetic trees were constructed for gapA, gyrB, rpoD and gltA genes individually, as well as for their concatenated sequences using the maximum likehood method with (1,000 bootstrap replicates) in MEGA6 software. The bacterial isolates clustered closely with P. Asiatica (Fig.S1). The pathogenicity assay was conducted on ginger plants at the 4-5 leaves stage. Ten plantlets were injected with 500 μl of bacterial suspension (107 to 108 CFU/ml) at the stem base using a sterile syringe. An equal number of plantlets were inoculated with sterile water as control. All plants were maintained at 30 °C, 80% relative humidity (RH) and a 16-h light/8-h dark period cycle. In addition, healthy rhizomes were surface-sterilized with 1% NaClO for 1 min, rinsed with sterile water, and then cut into segments. Ten rhizome segments were inoculated with 1 ml bacterial suspension and incubated in a climate-controlled incubator at 30°C, 80% relative humidity (RH) and in complete darkness. Pathogenicity assays were conducted in three independent replicates. After 2-3 weeks post inoculation, control plantlets and rhizome segments remained asymptomatic. In contrast, inoculated plantlets developed chlorosis, exhibited leaf yellowing and necrosis, consistent with field observation (Fig. S2). Concurrently, the treated plants displayed water-soaked lesions and stem base rot. Infected rhizomes developed severe symptoms including water-soaking, browning, and tissue decay. Bacterial colonies were successfully re-isolated from the symptomatic leaves, stem and rhizome, and identified through morphological and molecular characterization, fulfilling Koch’s postulates. Pseudomonas asiatica has been previously reported as a pathogen causing mastitis in cows (Sultana et al., 2025), this study represents the first documented case of P. asiatica induced rhizome rot in ginger in China. These findings provide a critical foundation for developing targeted disease management strategies against this emerging ginger pathogen.